Isoprenoids are ubiquitous in nature. They comprise a diverse family of over 40,000 individual products, many of which are vital to living organisms. Isoprenoids serve to maintain cellular fluidity, electron transport, and other metabolic functions. A vast number of natural and synthetic isoprenoids are useful as pharmaceuticals, cosmetics, perfumes, pigments and colorants, fungicides, antiseptics, nutraceuticals, and fine chemical intermediates.
An isoprenoid product is typically composed of repeating five carbon isopentenyl diphosphate (IPP) units, although irregular isoprenoids and polyterpenes have been reported. In nature, isoprenoids are synthesized by consecutive condensations of their precursor IPP and its isomer dimethylallyl pyrophosphate (DMAPP). Two pathways for these precursors are known. Eukaryotes, with the exception of plants, generally use the mevalonate-dependent (MEV) pathway to convert acetyl coenzyme A (acetyl-CoA) to IPP, which is subsequently isomerized to DMAPP. Prokaryotes, with some exceptions, typically employ only the mevalonate-independent or deoxyxylulose-5-phosphate (DXP) pathway to produce IPP and DMAPP. Plants use both the MEV pathway and the DXP pathway. See Rohmer et al. (1993) Biochem. J. 295:517-524; Lange et al. (2000) Proc. Natl. Acad. Sci. USA 97(24):13172-13177; Rohdich et al. (2002) Proc. Natl. Acad. Sci. USA 99:1158-1163.
Traditionally, isoprenoids have been manufactured by extraction from natural sources such as plants, microbes, and animals. However, the yield by way of extraction is usually very low due to a number of profound limitations. First, most isoprenoids accumulate in nature in only small amounts. Second, the source organisms in general are not amenable to the large-scale cultivation that is necessary to produce commercially viable quantities of a desired isoprenoid. Third, the requirement of certain toxic solvents for isoprenoid extraction necessitates special handling and disposal procedures, and thus complicating the commercial production of isoprenoids.
The elucidation of the MEV and DXP metabolic pathways has made biosynthetic production of isoprenoids feasible For instance, microbes have been engineered to overexpress a part of or the entire mevalonate pathway for production of an isoprenoid named amorpha-4,11-diene (U.S. Pat. Nos. 7,172,886 and 7,192,751) Other efforts have focused on balancing the pool of glyceraldehyde-3-phosphate and pyruvate, or on increasing the expression of 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and IPP isomerase (idi). See Farmer et al. (2001) Biotechnol. Prog. 17:57-61; Kajiwara et al. (1997) Biochem. J. 324:421-426; and Kim et al. (2001) Biotechnol. Bioeng. 72:408-415. Nevertheless, the conventional methods for monitoring and quantitation of metabolite levels in cells that utilize the mevalonate pathway suffer from a number of profound drawbacks. First, the conventional methods can only monitor a very limited number of the cofactors and metabolites involved in the mevalonate pathway. Second, this limitation is exacerbated by the fact that metabolites and individual cofactors of the pathway exhibit varying chemical stability profiles, which has made extraction and quantitation of more than several of such metabolites and cofactors difficult to achieve. There thus remains a need for methods and compositions useful for monitoring metabolic pathways such as the mevalonate pathway. The present invention addresses this need and provides related advantages as well.